Gene regulation is fundamental to the problems of cell differentiation and tissue morphogenesis, including cancer. Neurospora crassa possesses a well-developed genetic system, including DNA transformation. These have enabled extensive genetic analysis of several regulated N. crassa genes, providing detailed models of transcriptional regulation for more complex eukaryotic systems. Recently I developed an RNA polymerase II in vitro transcription system for N. crassa, enabling biochemical analysis to be extended to fungal transcriptional regulation for the first time. The aim of this project is to identify and functionally characterise the basic protein and DNA components of the N. crassa RNA polymerase II transcription apparatus, in order to provide a framework for defining biochemically how genetically characterized fungal regulatory proteins modulate transcription. Specifically, the aims of the project will be: (i) Using the strongly expressed 'housekeeping' gene, am (glutamate dehydrogenase) as a model, N. crassa RNA polymerase II transcription factors will be isolated from the existing crude in vitro system. The factors will be identified and then assayed during isolation by their binding to specific DNA sequences and/or by their requirement in a transcription assay containing separated protein fractions. (ii) The promoter DNA sequences required for am transcription will be determined. These sequences will be identified by their interaction with specific transcription factors, and by creating systematic sets of promoter mutations whose effects will be tested using in vivo and in vitro transcription assays. (iii) The different steps in the formation of an N. crassa RNA polymerase II transcription initiation complex, and the factors required for each step, will be determined by order-of-addition experiments, and by using inhibitors of specific steps in transcription initiation. (iv) The possibility of developing in vitro transcription systems for genes from other fungi such as S. cerevisiae, S. pombe and A. nidulans will be explored using N. crassa transcription factors either alone or in combination with proteins from these other fungi.